Live-imaging of oxygen radical formation in ex-vivo gills after exposure to anoxia and anoxia-reoxygenation: physiological consequences for a hypoxia tolerant bivalve

GEORGINA RIVERA-INGRAHAM; IARA ROCCHETTA; ULF BICKMEYER; DORIS ABELE

Congreso; PHYSIOMAR 14 International Meeting; 2014

Intertidal blue mussels, Mytilus edulis, experience hypoxia reoxygenation during tidal emersion and resubmersion cycles, and this is often suggested to represent a major stress for the animals, especially for their respiratory tissues, the gills. In a first approach, we applied live-imaging techniques to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue under normoxic conditions. Our results indicate that in the ciliated epithelial cells composing the gill filaments, two mitochondrial clusters with distinct membrane potential (Δψm) (JC-1 staining) coexist, where cilia-associated mitochondria show the weakest polarization. Along average gill filaments, O2?- (DHE staining, 2-OH-E+ fluorescence) concentrates in the outermost areas of the ciliated epithelial cells composing the filaments, which additionally concentrates the highest mitochondrial densities (MTK Deep Red 633 staining). On the other hand, H2O2, HOO? and ONOO- radicals (assessed through C-H2DFFDA staining, DCF fluorescence) are mainly formed within the blood sinus of the gill filaments and are likely to be produced by hemocytes as defense against invading pathogens.   In a second approach, we exposed mussels to experimental short (48h) and prolonged (72h) anoxia and subsequent reoxygenation and analyzed the respiratory response in excised gill tissue and the effects of treatment on ROS and RNS formation. Our aim was to understand if this ?natural stress? would indeed produce oxidative damage and whether antioxidant defenses are induced under anoxia, to prevent oxidative damage during reoxygenation. Exposure to declining pO2 in the respiration chamber caused an increase of gill metabolic rate between 21 and 10 kPa, a pO2 range in which whole animal respiration is reported to be oxyregulating. Exposure of the animals to severe anoxia caused an onset of anaerobiosis (succinate accumulation) and shifted high and low critical pc values (pc1: onset of oxyregulation in gills, pc2: switch from oxyregulation to oxyconformity) to higher pO2. Both 2-OH-E+ and DCF fluorescence decreased strongly during anoxic exposure of the mussels and increased upon reoxygenation. This ROS/RNS burst induced lipid peroxidation in the mantle, but neither were protein carbonyl levels increased (oxidative damage in the protein fraction), nor did the tissue glutathione concentration change in the gills. Further, analysis of apoptosis markers indicated no induction of cell death in the gills. To our knowledge, this is the first work that directly measures ROS/RNS formation ex-vivo during anoxia reoxygenation in gills isolated from mussels.   Our results open new perspectives for unraveling the different ecophysiological roles of ROS in gills and we conclude that hypoxia tolerant intertidal mussels do not suffer major oxidative stress in gill and mantle tissues under these experimental conditions.